Abstract

ICP27 is an HSV-1 immediate early protein required for the switch from early to late gene expression. ICP27 binds viral and cellular RNAs, and recently a yeast three-hybrid (Y3-H) analysis has identified an array of viral RNA sequences that interact with ICP27.

Presented here are analyses of functional assays using a selection of the Y3-H identified RNA sequences inserted into the 5’ untranslated region (UTR) of a chloramphenicol acetyl transferase (CAT) reporter plasmid. A set of plasmids was transfected into baby hamster kidney (BHK) cells and CAT assays carried out to analyse the effects of the sequences on gene expression. Results indicated that expression was increased when ICP27-binding sequences were present even though no viral proteins were present. Comparison of sequences revealed that no common activation code or RNA structure was present that could be responsible for the increase in CAT gene expression. The levels of expression were further determined in the presence of wild type (wt), ICP27-null or ICP27 mutant HSV-1 infection to investigate whether ICP27 had any affect on CAT expression when ICP27-binding sequences were present. Interestingly, enhanced expression was observed during wt HSV-1-infection when ICP27-binding sequences were present, whereas little to no enhancement was observed during ICP27-null or mutant virus infections. However, a higher fold increase in CAT gene expression was observed during a HSV-1 infection when ICP27-binding sequences were not present. This indicated that an inhibitory effect on CAT expression observed during wt HSV-1 infection when ICP27-binding sequences were present was ICP27-depdnent. As a control, a noncoding, protein binding regulatory HPV RNA sequence was inserted into the CAT reporter plasmid, transfected into BHK cells and then infected with wt HSV-1, ICP27-null or ICP27 mutant viruses. Surprisingly, CAT expression was increased, albeit to only a limited extent, indicating that the previously observed increase in gene expression was not HSV-1 sequence specific. However, upon transfection of plasmids with the HPV control sequence inserted in the reverse orientation and a subsequent infection of cells no increase in CAT expression was observed.

Analysis of the control sequence in the reverse orientation identified a shortage in G residues, which led to the construction of CAT reporter plasmids containing homopolymer sequences to inserted into the 5’UTR. A series of transfections and subsequent mock, wt HSV-1 or ICP27-null virus infections were carried out using this set of constructs. CAT assay analysis revealed an increase in CAT expression, to levels similar to those observed when the HSV-1 sequences were present, when poly(G) homopolymers were used as inserts during wt HSV-1 infection, whereas poly(A), (C) and (T) gave low levels of expression.